Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters
Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters
复制标题
Chryseobacter sp. ChKRED20 的结构引导工程。
DOI：
10.1016/j.enzmictec.2019.03.001
复制
发表时间：
2019-06-01
影响因子：
3.4
通讯作者：
Wu, Zhong-Liu
中科院分区：
文献类型：
--
作者：
Li, Tong-Biao;Zhao, Feng-Jiao;Wu, Zhong-Liu
ChKRED20是一种从金黄杆菌属（Chryseobacterium sp.）CA49的基因组中鉴定出的稳健的依赖NADH的酮还原酶，它可以使用2 - 丙醇作为最终还原剂。对于一些药物相关底物，野生型能够还原超过100克/升的酮，显示出在工业应用方面的显著潜力。在这项工作中，为了克服ChKRED20对芳基酮酯的局限性，我们首先将ChKRED20/NAD(+)复合物的X射线晶体结构精修至1.6埃的分辨率，然后在关键氨基酸位点进行三轮迭代饱和诱变以重塑酶的活性腔。对于2 - 氧代 - 2 - 苯基乙酸甲酯和3 - 氧代 - 3 - 苯基丙酸乙酯，获得了几个活性增强的突变体；对于2 - 氧代 - 4 - 苯基丁酸乙酯，获得了改进的突变体，其k(cat)/K - m增加到野生型的196倍。在100克/升的底物负载量下，所有三种底物都能被选定的ChKRED20突变体完全还原，并以>90%的分离产率和97 - >99%的对映体过量（ee）生成相应的手性醇。
ChKRED20 is a robust NADH-dependent ketoreductase identified from the genome of Chryseobacterium sp. CA49 that can use 2-propanol as the ultimate reducing agent. The wild-type can reduce over 100 g/l ketones for some pharmaceutical relevant substrates, exhibiting a remarkable potential for industrial application. In this work, to overcome the limitation of ChKRED20 to aryl ketoesters, we first refined the X-ray crystal structure of ChKRED20/NAD(+) complex at a resolution of 1.6 angstrom, and then performed three rounds of iterative saturation mutagenesis at critical amino acid sites to reshape the active cavity of the enzyme. For methyl 2-oxo-2-phenylacetate and ethyl 3-oxo-3-phenylpropanoate, several gain-of-activity mutants were achieved, and for ethyl 2-oxo-4-phenylbutanoate, improved mutants were achieved with k(cat)/K-m increasing to 196-fold of the wild-type. All three substrates were completely reduced at 100 g/l loading catalyzed with selected ChKRED20 mutants, and deliver the corresponding chiral alcohols with > 90% isolated yield and 97 - > 99%ee.